The biological functions of many RNAs depend on compact tertiary structures with irregular backbone conformations and close juxtapositions of phosphates. The resulting unfavorable electrostatic free energy is a major barrier to folding, and causes RNA tertiary structure to be much more sensitive to the ion and solvent environment than is secondary structure. The long term goal of these studies is to provide a systematic and quantitative picture of ion and solvent interactions stabilizing RNA tertiary structures, and relate the picture to the underlying electrostatic properties of RNA. A major aim of the proposed studies is to obtain accurate Mg 2+ - RNA binding isotherms for a set of RNA structures that illustrate the full range of potential Mg 2+ binding environments, including RNAs stabilized entirely by fully hydrated Mg 2+, RNAs in which Mg(H20)6[2]+ may be hydrogen bonded to pockets or channels in the RNA structure, RNAs with highly dehydrated ions bound atspecific sites, and RNAs that mimic the close packing of helices in large RNAs such as the ribosome. Mg 2+ binding to RNAs trapped in partially folded states (secondary structure only) will also be studied, to provide a picture of the net change in Mg 2+ binding during the folding reaction. Newly developed fluorescence-based methods for directly measuring free and RNA-bound Mg 2+ will be used in these studies. Other studies will look at the proposed phenomenon of "electrostatic collapse" as a separate, Mg 2+- dependent transition in folding of large RNAs, at the selectivity of RNAs for monovatent ions, which may be a widespread feature of compact RNAs and reflect partial dehydration of monovalent ions bound close to the RNA surface, and at binding of small peptides or proteins that may serve as probes of ions and hydrating water stabilizing an RNA structure. Lastly, these results will be used as benchmarks for comparison with theoretical predictions of ion - RNA interactions, particularly a recently developed framework that uses the non-linear Poisson-Boltzmann equation to describe hydrated RNA-bound Mg 2+ and a Born model to compute the energetics of partially dehydrated ions at specific locations in the RNA. The ability to predict the Mg2+-dependent folding free energies of RNA tertiary conformations would be useful in a number of contexts.